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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous 79, 86 Rabbit IgG

Western blot analysis of extracts from IFN-alpha treated Jurkat cells and HeLa cells (left), as well as EGF treated A431 cells (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb. Note that the basal phospho-Stat3 in A431 is detected by the antibody.

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Immunohistochemical analysis of paraffin-embedded Apc (min/+) mouse intestine, using Phosho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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Immunohistochemical analysis of paraffin embedded human breast carcinoma, specifically endothelial cells, untreated (left) or lambda phosphatase treated (right), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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Immunnohistochemical analysis of paraffin-embedded human lung carcinoma, showing nuclear localization, using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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Immunohistochemical analysis using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb on SignalSlide® Phospho-Stat1/3/5 IHC Controls #8105 (paraffin-embedded HeLa cell pellets, untreated (left), treated with Human Interferon-α1 (hIFN-α1) #8927 (middle), or treated with Human Epidermal Growth Factor (hEGF) #8916 (right).

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Immunohistochemical analysis of frozen H1650 xenograft using Phospho-Stat3 (Tyr705)(D3A7) XP® Rabbit mAb.

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Confocal immunofluorescent analysis of HeLa cells, IFN-alpha treated (left) or untreated (right), labeled with Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb (green).

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Flow cytometric analysis of HeLa cells, untreated (blue) or IFN-α treated (green), using Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb.

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Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 Hep G2 cells starved overnight and treated with IL-6 (100 ng/ml) for 30 minutes, and either 10 μl of Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human IRF-1 promoter primers, SimpleChIP® Human c-Fos Promoter Primers #4663, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#7720, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

protocol id: 10

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 0.76 g Tris base, 2 g SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H2O. Adjust pH to 6.8 with HCl.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

Page

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.

    NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

  3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
  4. Protein A Magnetic Beads: Use Protein A (#8687) for rabbit IgG immunoprecipitation.
  5. 6-Tube Magnetic Separation Rack: (#7017).
  6. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix.
  7. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

  1. Vortex to mix beads.
  2. Add 10–30 µl of 50% Protein A magnetic bead slurry of to 200 µl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30–60 min.
  4. Pellet beads using magnetic separation rack. Transfer the supernatant to a fresh tube.
  5. Proceed to immunoprecipitation below.

Immunoprecipitation

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Add protein A magnetic beads (10–30 µl of 50% bead slurry). Incubate with rotation for 10–30 min at 4°C.
  3. Pellet beads using magnetic separation rack. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to analyze by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 µl) on SDS-PAGE (4–20%).

posted December 2008

revised November 2013

protocol id: 410

Page

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. Antigen Unmasking EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C10H14N2O8Na2•2H2O) to 1 L dH2O. Adjust pH to 8.0.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: Vector® NovaRED™ (Vector Laboratories).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0: follow with 15 min at a sub-boiling temperature. No cooling is necessary.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Prepare Vector® NovaRED™ per manufacturer's recommendations.
  12. Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin per manufacturer’s instructions.
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips.

posted February 2010

revised November 2013

protocol id: 304

Immunohistochemistry (Frozen)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol (anhydrous denatured, histological grade 100% and 95%).
  3. Hematoxylin (optional).
  4. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  5. Fixative: 3% formaldehyde.
    1. To prepare 100 ml, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS.
  6. Wash Buffer: 1X Tris Buffered Saline (TBS).

    To prepare 1L 1X TBS add 100 ml 10X Tris Buffered Saline (#12498) to 900 ml dH2O, mix.

  7. Methanol/Peroxidase: To prepare, add 10 ml 30% H2O2 to 90 ml methanol. Store at -20°C.
  8. Blocking Solution: 1X TBS/0.3% Triton™ X-100/5% Normal Goat Serum (#5425). To prepare, add 500 µl goat serum and 30 µl Triton™ X-100 to 9.5 ml 1X TBS.
  9. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  10. Substrate: Vector® NovaRED™ (Vector Laboratories).

B. Sectioning

  1. For tissue stored at -80°C: Remove from freezer and equilibrate at -20°C for approximately 15 min before attempting to section. This may prevent cracking of the block when sectioning.
  2. Section tissue at a range of 6–8 µm and place on positively charged slides.
  3. Allow sections to air dry on bench for a few min before fixing (this helps sections adhere to slides).

C. Fixation

  1. Fix sections in 3% formaldehyde for 15 min at room temperature. Proceed with staining procedure immediately (Section D).

D. Staining

  1. Wash sections in wash buffer two times for 5 min.
  2. Incubate for 10 min at room temperature in methanol/peroxidase.
  3. Wash sections in wash buffer two times for 5 min.
  4. Block each section with 100–400 µl blocking solution for 1 hr at room temperature.
  5. Remove blocking solution and add 100–400 µl primary antibody diluted in blocking solution to each section.
  6. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections in wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Prepare Vector® NovaRED™ (Vector Laboratories) per manufacturer's instructions.
  12. Apply 100-400 µl substrate to each section and monitor staining closely. 5-15 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin per manufacturer’s instructions.
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips.

posted February 2010

revised November 2013

protocol id: 323

Page

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Methanol, 100%
  4. Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
  5. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  6. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

  7. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
    NOTE: Formaldehyde is toxic, use only in fume hood.
  2. Allow cells to fix for 15 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Methanol Permeabilization Step: Cover cells with ice-cold 100% methanol (use enough to cover completely to a depth of 3–5 mm, DO NOT LET DRY), incubate in methanol for 10 minutes at –20°C, rinse in 1X PBS for 5 minutes.
  2. Block specimen in Blocking Buffer for 60 minutes.
  3. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  4. Aspirate blocking solution, apply diluted primary antibody.
  5. Incubate overnight at 4°C.
  6. Rinse three times in 1X PBS for 5 minutes each.
  7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  8. Rinse in 1X PBS as in step 6.
  9. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  10. For best results examine specimens immediately using appropriate excitation wavelength. For long term storage, store slides flat at 4°C protected from light.

posted November 2006

revised December 2010

protocol id: 32

Page

Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  5. Recommended Anti-Rabbit secondary antibodies:

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 10 min at 37°C.
  4. Chill tubes on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
  3. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in 2–3 ml incubation buffer.
  6. Resuspend cells in fluorochrome-conjugated secondary antibody, diluted in incubation buffer at the recommended dilution.
  7. Incubate for 30 min at room temperature.
  8. Wash by centrifugation in 2–3 ml incubation buffer.
  9. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised September 2013

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
  6. Secondary Antibodies:

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 30–60 min at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in incubation buffer according to the manufacturer’s recommendations.
  6. Incubate for 30 min at room temperature.
  7. Wash by centrifugation in 2–3 ml incubation buffer.
  8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

protocol id: 404

Page

Chromatin IP

Specific for product: SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005.

Required Reagents

Reagents Included:

  1. Glycine Solution (10X)
  2. Buffer A (4X)
  3. Buffer B (4X)
  4. ChIP Buffer (10X)
  5. ChIP Elution Buffer (2X)
  6. 5 M NaCl
  7. 0.5 M EDTA
  8. 1M DTT
  9. DNA Binding Reagent A (add 12 ml isopropanol before use)
  10. DNA Wash Reagent B (add 24 ml ethanol before use)
  11. DNA Elution Reagent C
  12. DNA Spin Columns
  13. Protease Inhibitor Cocktail (200X)
  14. RNAse A (10 mg/ml)
  15. Micrococcal Nuclease (2000 gel units/µl)
  16. Proteinase K (20 mg/ml)
  17. SimpleChIP® Human RPL30 Exon 3 Primers #7014
  18. SimpleChIP® Mouse RPL30 Intron 2 Primers #7015
  19. Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated) #4620
  20. Normal Rabbit IgG #2729
  21. ChIP-Grade Protein G Magnetic Beads #9006

Reagents Not Included:

  1. 6-Tube Magnetic Separation Rack #7017
  2. Formaldehyde (37%)
  3. Ethanol (96-100%)
  4. Isopropanol
  5. 1X PBS
  6. Nuclease-free water
  7. Taq DNA polymerase
  8. dNTP Mix

I. Tissue Cross-linking and Sample Preparation:

When harvesting tissue, remove unwanted material such as fat and necrotic material from the sample. Tissue can then be processed and cross-linked immediately, or frozen on dry ice for processing later. For optimal chromatin yield and ChIP results, use 25 mg of tissue for each immunoprecipitation to be performed. The chromatin yield does vary between tissue types and some tissues may require more than 25 mg for each immunoprecipitation. Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue. One additional chromatin sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV).

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
  • Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 μl 200X PIC per 25 mg of tissue to be processed and place on ice.
  • Prepare 45 μl of 37% formaldehyde per 25 mg of tissue to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer’s expiration date.

A. Cross-linking

  1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be performed.
  2. Place tissue sample in a 60 mm or 100 mm dish and finely mince using a clean scalpel or razor blade. Keep dish on ice. It is important to keep the tissue cold to avoid protein degradation.
  3. Transfer minced tissue to a 15 ml conical tube.
  4. Add 1 ml of PBS + PIC per 25 mg tissue to the conical tube.
  5. To crosslink proteins to DNA, add 45 μl of 37% formaldehyde per 1 ml of PBS + PIC and rock at room temp for 20 min. Final formaldehyde concentration is 1.5%.
  6. Stop cross-linking by adding 100 μl of 10x glycine per 1 ml of PBS + PIC and mix for 5 min at room temperature.
  7. Centrifuge tissue at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  8. Remove supernatant and wash one time with 1 ml PBS + PIC per 25 mg tissue.
  9. Repeat centrifugation at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  10. Remove supernatant and resuspend tissue in 1 ml PBS + PIC per 25 mg tissue and store on ice. Disaggregate tissue into single-cell suspension using a Medimachine (Part B) or Dounce homogenizer (Part C).

B. Tissue Disaggregation Using Medimachine from BD Biosciences (part #340587)

  1. Cut off the end of a 1000 μL pipette tip to enlarge the opening for transfer of tissue chunks.
  2. Transfer 1 ml of tissue resuspended in PBS + PIC into the top chamber of a 50 mm medicone (part #340592).
  3. Grind tissue for 2 min according to manufacturer’s instructions.
  4. Collect cell suspension from the bottom chamber of the medicone using a 1 ml syringe and 18 gauge blunt needle. Transfer cell suspension to a 15 ml conical tube and place on ice.
  5. Repeat steps 2 to 4 until all the tissue is processed into a homogenous suspension.
  6. If more grinding is necessary, add more PBS + PIC to tissue. Repeat steps 2 to 5 until all tissue is ground into a homogeneous suspension.
  7. Check for single-cell suspension by microscope (optional).
  8. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  9. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

C. Tissue Disaggregation Using a Dounce Homogenizer:

  1. Transfer tissue resuspended in PBS + PIC to a Dounce homogenizer.
  2. Disaggregate tissue pieces with 20-25 strokes. Check for single-cell suspension by microscope (optional).
  3. Transfer cell suspension to a 15 ml conical tube and centrifuge at 1,500 rpm in a bench top centrifuge for 5 min at 4°C.
  4. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

II. Cell Culture Cross-linking and Sample Preparation:

For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to be performed. For HeLa cells, this is equivalent to half of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. One additional sample should be processed for Analysis of Chromatin Digestion and Concentration (Section IV). Include one extra dish of cells in experiment to be used for determination of cell number using a hemocytometer.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed.
  • Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 μl 200X PIC per 15 cm dish to be processed and place on ice.
  • Prepare 40 ml of PBS per 15 cm dish to be processed and place on ice.
  • Prepare 540 μl of 37% formaldehyde per 15 cm dish of cells to be processed and keep at room temperature. Use fresh formaldehyde that is not past the manufacturer’s expiration date.
  1. To crosslink proteins to DNA, add 540 μl of 37% formaldehyde to each 15 cm culture dish containing 20 ml medium. Swirl briefly to mix and incubate 10 min at room temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may result in a color change of the medium.
  2. Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min at room temperature. Addition of glycine may result in a color change of the medium.
  3. For suspension cells, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm in a bench top centrifuge 5 min at 4°C and wash pellet two times with 20 ml ice-cold PBS. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).
  4. For adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from culture dish each time.
  5. Add 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer. Combine cells from all culture dishes into one 15 ml conical tube.
  6. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C. Remove supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

III. Nuclei Preparation and Chromatin Digestion:

One immunoprecipitation preparation (IP prep) is defined as 25 mg of disaggregated tissue or 4 x 106 tissue culture cells.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 1 M DTT. Make sure both are completely thawed and DTT crystals are completely in solution.
  • Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution.
  • Prepare 1 ml 1X Buffer A (250 μl 4X Buffer A + 750 μl water) + 0.5 μl 1M DTT + 5 μl 200X PIC per IP prep and place on ice.
  • Prepare 1.1 ml 1X Buffer B (275 μl 4X Buffer B + 825 μl water) + 0.55 μl 1M DTT per IP prep and place on ice.
  • Prepare 100 μl 1X ChIP Buffer (10 μl 10X ChIP Buffer + 90 μl water) + 0.5 μl 200X PIC per IP prep and place on ice.
  1. Resuspend cells in 1 ml ice-cold Buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 3 min.
  2. Pellet nuclei by centrifugation at 3,000 rpm in a bench top centrifuge for 5 min at 4°C. Remove supernatant and resuspend pellet in 1 ml ice-cold Buffer B + DTT per IP prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 μl Buffer B + DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per tube.
  3. Add 0.5 μl of Micrococcal Nuclease per IP prep, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and cell lines (see Appendix B). HeLa nuclei digested with 0.5 μl Micrococcal Nuclease per 4 x 106 cells and mouse liver tissue digested with 0.5 μl Micrococcal Nuclease per 25 mg of tissue gave the appropriate length DNA fragments (see Figure 3).
  4. Stop digest by adding 10 μl of 0.5 M EDTA per IP prep and placing tube on ice.
  5. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
  6. Resuspend nuclear pellet in 100 μl of 1X ChIP buffer + PIC per IP prep and incubate on ice for 10 min.
  7. Sonicate up to 500 μl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  8. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
  9. Transfer supernatant to a new tube. This is the cross-linked chromatin preparation, which should be stored at -80°C until further use. Remove 50 μl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration (Section IV).

IV. Analysis of Chromatin Digestion and Concentration (Recommended Step)

  1. To the 50 μl chromatin sample (from Step 9 in Section III), add 100 μl nuclease-free water, 6 μl 5 M NaCl, and 2 μl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
  2. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate samples at 65°C for 2 h.
  3. Purify DNA from samples using DNA purification spin columns as described in Section VII.
  4. After purification of DNA, remove a 10 μl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be digested to a length of approximately 150-900 bp (1 to 5 nucleosomes; see Figure 3).
  5. To determine DNA concentration, transfer 2 μl of purified DNA to 98 μl nuclease-free water to give a 50-fold dilution and read the OD260. The concentration of DNA in μg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 200 μg/ml.

NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion of chromatin may diminish signal in the PCR quantification. Under-digestion of chromatin may lead to increased background signal and lower resolution. Adding too little chromatin to the IP may result in diminished signal in the PCR quantification. A protocol for optimization of chromatin digestion can be found in Appendix B.

V. Chromatin Immunoprecipitation:

For optimal ChIP results, use approximately 5 to 10 μg of digested, cross-linked chromatin (as determined in Section IV) per immunoprecipitation. This should be roughly equivalent to a single 100 μl IP prep from 25 mg of disaggregated tissue or 4 x 106 tissue culture cells. Typically, 100 μl of digested chromatin is diluted into 400 μl 1X ChIP Buffer prior to the addition of antibodies. However, if more than 100 μl of chromatin is required per IP, the cross-linked chromatin preparation does not need to be diluted as described below. Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes.

Before starting:

  • Remove and warm 200X Protease Inhibitor Cocktail (PIC). Make sure PIC is completely thawed.
  • Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution.
  • Thaw digested chromatin preparation (from Step 9 in Section III) and place on ice.
  • Prepare low salt wash: 3 ml 1X ChIP Buffer (300 μl 10X ChIP Buffer + 2.7 ml water) per immunoprecipitation. Store at room temperature until use.
  • Prepare high salt wash: 1 ml 1X ChIP Buffer (100 μl 10X ChIP Buffer + 900 μl water) + 70 μl 5M NaCl per immunoprecipitation. Store at room temperature until use.
  1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin into the desired number of immunoprecipitations: 400 μl of 1X ChIP Buffer (40 μl of 10X ChIP Buffer + 360 μl water) + 2 μl 200X PIC per immunoprecipitation. When determining the number of immunoprecipitations, remember to include the positive control Histone H3 (D2B12) XP® Rabbit mAb #4620 and negative control Normal Rabbit IgG antibody samples. Place mix on ice.
  2. To the prepared 1X ChIP buffer, add the equivalent of 100 μl (5 to 10 μg of chromatin) of the digested, cross-linked chromatin preparation (from Step 9 in Section III) per immunoprecipitation. For example, for 10 immunoprecipitations, prepare a tube containing 4 ml 1X ChIP Buffer (400 μl 10X ChIP Buffer + 3.6 ml water) + 20 μl 200X PIC + 1 ml digested chromatin preparation.
  3. Remove a 10 μl sample of the diluted chromatin and transfer to a microfuge tube. This is your 2% Input Sample, which can be stored at -20°C until further use (Step 1 in Section VI).
  4. For each immunoprecipitation, transfer 500 μl of the diluted chromatin to a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody required per IP varies and should be determined by the user. For the positive control Histone H3 (D2B12) XP® Rabbit mAb #4620, add 10 μl to the IP sample. For the negative control Normal Rabbit IgG, add 1 μl (1 μg) to 2 μl (2 μg) to the IP sample. Incubate IP samples 4 h to overnight at 4°C with rotation.
  5. Resuspend ChIP-Grade Protein G Magnetic Beads by gently vortexing. Immediately add 30 μl of Protein G Magnetic Beads to each IP reaction and incubate for 2 h at 4°C with rotation.
  6. Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in a Magnetic Separation Rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant.
  7. Wash Protein G Magnetic Beads by adding 1 ml of low salt wash to the beads and incubate at 4°C for 5 min with rotation. Repeat steps 6 and 7 two additional times for a total of 3 low salt washes.
  8. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation.
  9. Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in a Magnetic Separation Rack. Wait 1 to 2 min for solution to clear and then carefully remove supernatant. Immediately proceed to Section VI.

VI. Elution of Chromatin from Antibody/Protein G Magnetic Beads and Reversal of Cross-links:

Before starting:

  • Remove and warm 2X ChIP Elution Buffer in a 37°C water bath and ensure SDS is in solution.
  • Set a water bath or thermomixer to 65°C.
  • Prepare 150 μl 1X ChIP Elution Buffer (75 μl 2X ChIP Elution Buffer + 75 μl water) for each immunoprecipitation and the 2% input sample.
  1. Add 150 μl of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at room temperature until Step 6.
  2. Add 150 μl 1X ChIP Elution Buffer to each IP sample.
  3. Elute chromatin from the antibody/Protein G Magnetic Beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works best for this step. Alternatively, elutions can be performed at room temperature with rotation, but may not be as complete.
  4. Pellet Protein G Magnetic Beads by placing the tubes in a Magnetic Separation Rack and wait 1 to 2 min for solution to clear.
  5. Carefully transfer eluted chromatin supernatant to a new tube.
  6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 μl 5M NaCl and 2 μl Proteinase K, and incubate 2 h at 65°C. This incubation can be extended overnight.
  7. Immediately proceed to Section VII. Alternatively, samples can be stored at -20°C. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Reagent A (Section VII, Step 1).

VII. DNA Purification Using Spin Columns:

Before starting:

  • Add 12 ml of isopropanol to DNA Binding Reagent A and 24 ml of ethanol (96-100%) to DNA Wash Reagent B before use. These steps only have to be performed once prior to the first set of DNA purifications.
  • Remove one DNA purification spin column and collection tube for each DNA sample from Section VI.
  1. Add 600 μl DNA Binding Reagent A to each DNA sample and vortex briefly.
    • 4 volumes of DNA Binding Reagent A should be used for every 1 volume of sample.
  2. Transfer 375 μl of each sample from Step 1 to a DNA purification spin column in collection tube.
  3. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  4. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  5. Transfer the remaining 375 μl of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
  6. Add 700 μl of DNA Wash Reagent B to the spin column in collection tube.
  7. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  8. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  9. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
  10. Discard collection tube and liquid. Retain spin column.
  11. Add 50 μl of DNA Elution Reagent C to each spin column and place into a clean 1.5 ml microcentrifuge tube.
  12. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec to elute DNA.
  13. Remove and discard DNA purification spin column. Eluate is now purified DNA. Samples can be stored at -20°C.

VIII. Quantification of DNA by PCR:

Recommendations:

  • Use Filter-tip pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for the human or mouse RPL30 gene and can be used for either standard PCR or quantitative real-time PCR. If the user is performing ChIPs from another species, it is recommended that the user design the appropriate specific primers to DNA and determine the optimal PCR conditions.
  • A Hot-Start Taq polymerase is recommended to minimize the risk of non-specific PCR products.
  • PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:
Primer length: 24 nucleotides
Optimum Tm: 60°C
Optimum GC: 50%
Amplicon Size: 150 to 200 bp (for standard PCR)
80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method:

  1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 2% input sample, the positive control histone H3 sample, the negative control normal rabbit IgG sample, and a tube with no DNA to control for DNA contamination.
  2. Add 2 μl of the appropriate DNA sample to each tube.
  3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for loss of volume. Add 18 μl of master mix to each reaction tube.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free H2O 12.5 μl
10X PCR Buffer 2.0 μl
4 mM dNTP Mix 1.0 μl
5 µM RPL30 Primers 2.0 μl
Taq DNA Polymerase 0.5 μl
  1. Start the following PCR reaction program:
a. Initial Denaturation 95°C 5 min
b. Denature 95°C 30 sec
c. Anneal 62°C 30 sec
d. Extension 72°C 30 sec
e. Repeat Steps b-d for a total of 34 cycles.
f. Final Extension 72°C 5 min
  1. Remove 10 μl of each PCR product for analysis by 2% agarose gel or 10% poly-acrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR product is 161 bp for human RPL30 and 159 bp for mouse RPL30.

Real-Time Quantitative PCR Method:

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. PCR reactions should include the positive control histone H3 sample, the negative control normal rabbit IgG sample, a tube with no DNA to control for contamination, and a serial dilution of the 2% input chromatin DNA (undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of amplification.
  2. Add 2 μl of the appropriate DNA sample to each tube or well of the PCR plate.
  3. Prepare a master reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of reaction mix to each PCR reaction tube or well.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free H2O 6 μl
5 µM RPL30 Primers 2 μl
2X SYBR-Green Reaction Mix 10 μl
  1. Start the following PCR reaction program:
a. Initial Denaturation 95°C 3 min
b. Denature 95°C 15 sec
c. Anneal and Extension: 60°C 60 sec
d. Repeat steps b and c for a total of 40 cycles.
  1. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

Percent Input = 2% x 2(C[T] 2%Input Sample – C[T] IP Sample)

C[T] = CT = Threshold cycle of PCR reaction

APPENDIX A: Expected Chromatin Yield

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. The table to the right provides a range for the expected yield of chromatin from 25 mg of tissue compared to 4 x 106 HeLa cells, and the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP results, we recommend using 5 to 10 µg of digested, cross-linked chromatin per immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per each immunoprecipitation.

Tissue/Cell Total Chromatin Yield Expected DNA Concentration
Spleen 20-30 µg per 25 mg tissue 200-300 µg/ml
Liver 10-15 µg per 25 mg tissue 100-150 µg/ml
Kidney 8-10 µg per 25 mg tissue 80-100 µg/ml
Brain 2-5 µg per 25 mg tissue 20-50 µg/ml
Heart 2-5 µg per 25 mg tissue 20-50 µg/ml
HeLa 10-15 µg per 4 x 106 cells 100-150 µg/ml

APPENDIX B: Optimization of Chromatin Digestion

Optimal conditions for the digestion of cross-linked chromatin DNA to 150-900 base pairs in length is highly dependent on the ratio of Micrococcal Nuclease to the amount of tissue or number of cells used in the digest. Below is a protocol for determination of the optimal digestion conditions for a specific tissue or cell type.

  1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 107 cells (equivalent of 5 IP preps), as described in Sections I, II, and III. Stop after Step 2 of Section III and proceed as described below.
  2. Transfer 100 μl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice.
  3. Add 3 μl Micrococcal Nuclease stock to 27 μl of 1X Buffer B + DTT (1:10 dilution of enzyme).
  4. To each of the 5 tubes in Step 2, add 0 μl, 2.5 μl, 5 μl, 7.5 μl, or 10 μl of the diluted Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 min at 37°C with frequent mixing.
  5. Stop each digest by adding 10 μl of 0.5 M EDTA and placing tubes on ice.
  6. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant.
  7. Resuspend nuclear pellet in 200 μl of 1X ChIP buffer + PIC. Incubate on ice for 10 min.
  8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis may not be as complete.
  9. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C.
  10. Transfer 50 μl of each of the sonicated lysates to new microfuge tubes.
  11. To each 50 μl sample, add 100 μl nuclease-free water, 6 μl 5 M NaCl and 2 μl RNAse A. Vortex to mix and incubate samples at 37°C for 30 min.
  12. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 h.
  13. Remove 20 μl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker.
  14. Observe which of the digestion conditions produces DNA in the desired range of 150-900 base pairs (1 to 5 nucleosomes, see Figure 3). The volume of diluted Micrococcal Nuclease that produces the desired size of DNA fragments using this optimization protocol is equivalent to 10 times the volume of Micrococcal Nuclease stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 4 X 106 tissue culture cells) to produce the desired size of DNA fragments. For example, if 5 μl of diluted Micrococcal Nuclease produces DNA fragments of 150-900 base pairs in this protocol, then 0.5 μl of stock Micrococcal Nuclease should be added to one immunoprecipitation preparation during the digestion of chromatin in Section III.
  15. If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of Micrococcal Nuclease in each digest accordingly. Alternatively, the digestion time can be changed to increase or decrease the extent of DNA fragmentation.

APPENDIX C: Troubleshooting Guide

Problem Possible Causes Recommendation
1. Concentration of the digested chromatin is too low (low chromatin yield). Not enough tissue or cells were added to the chromatin digestion or cell nuclei were not completely lysed after digestion. Add additional chromatin to each IP to give at least 5 μg/IP and continue with protocol.
Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Some tissues may require processing of more than 25 mg per IP. The amount of tissue can be increased to 50 mg per IP, while still maintaining efficient chromatin fragmentation and extraction
Increase the number of sonications following chromatin digestion. Visualize cell nuclei under microscope before and after sonication to confirm complete lysis of nuclei.
2. Chromatin is under-digested and fragments are too large (greater than 900 bp). Large chromatin fragments can lead to increased background and lower resolution. Too many cells or not enough Micrococcal Nuclease was added to the chromatin digestion. Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Add less tissue or cells, or more Micrococcal nuclease to the chromatin digest. See Appendix B for optimization of chromatin digestion.
Tissue or cells may have been over cross-linked. Cross-linking for longer than 10 min may inhibit digestion of chromatin. Perform a time course at a fixed formaldehyde concentration. Shorten the time of cross-linking to 10 min or less.
3. Chromatin is over-digested and fragments are too small (exclusively 150 bp mono-nucleosome length). Complete digestion of chromatin to mono-nucleosome length DNA may diminish signal during PCR quantification, especially for amplicons greater than 150 bp in length. Not enough cells or too much Micrococcal Nuclease added to the chromatin digestion. Weigh tissue or count a separate plate of cells prior to cross-linking to determine accurate cell number. Add more tissue or cells, or less Micrococcal nuclease to the chromatin digest. See Appendix B for optimization of chromatin digestion.
4. No product or very little product in the input PCR reactions. Not enough DNA added to the PCR reaction or conditions are not optimal. Add more DNA to the PCR reaction or increase the number of amplification cycles.
PCR amplified region may span nucleosome-free region. Optimize the PCR conditions for experimental primer set using purified DNA from cross-linked and digested chromatin. Design a different primer set and decrease length of amplicon to less than 150 bp (see primer design recommendations in Section VIII).
Not enough chromatin added to the IP or chromatin is over-digested. For optimal ChIP results, add 5 to 10 μg chromatin per IP. See recommendations for problems 1 and 3 above.
5. No product in the positive control histone H3-IP RPL30 PCR reaction. Not enough chromatin or antibody added to the IP reaction or IP incubation time is too short. Be sure to add 5 to 10 μg of chromatin and 10 μl of antibody to each IP reaction and incubate with antibody over-night and an additional 2 h after adding Protein G beads.
Incomplete elution of chromatin from Protein G beads. Elution of chromatin from Protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.
6. Quantity of product in the negative control Rabbit IgG-IP and positive control histone H3-IP PCR reactions is equivalent (high background signal). Too much or not enough chromatin added to the IP reaction. Or too much antibody added to the IP reaction. For optimal ChIP results, add 5 to 10 µg of chromatin and 10 μl of histone H3 antibody to each IP reaction. Reduce the amount of normal rabbit IgG to 1 μl per IP.
Too much DNA added to the PCR reaction or too many cycles of amplification. Add less DNA to the PCR reaction or decrease the number of PCR cycles. It is very important that the PCR products are analyzed within the linear amplification phase of PCR. Otherwise, the differences in quantities of starting DNA cannot be accurately measured. Alternatively, quantify immunoprecipitations using real-time quantitative PCR.
7. No product in the Experimental Antibody-IP PCR reaction. Not enough DNA added to the PCR reaction. Add more DNA to the PCR reaction or increase the number of amplification cycles.
Not enough antibody added to the IP reaction. Typically a range of 1 to 5 μg of antibody are added to the IP reaction; however, the exact amount depends greatly on the individual antibody. Increase the amount of antibody added to the IP.
Antibody does not work for ChIP. Find an alternate antibody source.

posted December 2011

revised November 2013

protocol id: 82

Product Usage Information

Application Dilutions
Western Blotting 1:2000
Immunoprecipitation 1:100
Immunohistochemistry 1:400
Immunofluorescence 1:100
Flow Cytometry 1:200
Chromatin IP 1:100

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAb detects endogenous levels of Stat3 only when phosphorylated at tyrosine 705. This antibody does not cross-react with phospho-EGFR or the corresponding phospho-tyrosines of other Stat proteins.


Species Reactivity: Human, Mouse, Rat, Monkey
Species predicted to react based on 100% sequence homology: Hamster, Bovine, Pig, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr705 of mouse Stat3.

The Stat3 transcription factor is an important signaling molecule for many cytokines and growth factor receptors (1) and is required for murine fetal development (2). Research studies have shown that Stat3 is constitutively activated in a number of human tumors (3,4) and possesses oncogenic potential (5) and anti-apoptotic activities (3). Stat3 is activated by phosphorylation at Tyr705, which induces dimerization, nuclear translocation, and DNA binding (6,7). Transcriptional activation seems to be regulated by phosphorylation at Ser727 through the MAPK or mTOR pathways (8,9). Stat3 isoform expression appears to reflect biological function as the relative expression levels of Stat3α (86 kDa) and Stat3β (79 kDa) depend on cell type, ligand exposure, or cell maturation stage (10). It is notable that Stat3β lacks the serine phosphorylation site within the carboxy-terminal transcriptional activation domain (8).


1.  Heim, M.H. (2001) J Recept Signal Transduct Res 19, 75-120.

2.  Takeda, K. et al. (1997) Proc Natl Acad Sci U S A 94, 3801-4.

3.  Catlett-Falcone, R. et al. (1999) Immunity 10, 105-15.

4.  Garcia, R. and Jove, R. (1998) J Biomed Sci 5, 79-85.

5.  Bromberg, J.F. et al. (1999) Cell 98, 295-303.

6.  Darnell, J.E. et al. (1994) Science 264, 1415-21.

7.  Ihle, J.N. (1995) Nature 377, 591-4.

8.  Wen, Z. et al. (1995) Cell 82, 241-50.

9.  Yokogami, K. et al. (2000) Curr Biol 10, 47-50.

10.  Biethahn, S. et al. (1999) Exp Hematol 27, 885-94.


Entrez-Gene Id 6774
Swiss-Prot Acc. P40763


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
XP® is a trademark of Cell Signaling Technology, Inc.